Understanding the mechanism of DNA replication in human cells is a fundamental problem in biology and has direct relevance to cancer research. Toward this goal, the mechanism of DNA replication from the Simian Virus 40 (SV40) origin of DNA replication (ori) will be investigated. Using cell free extracts prepared from human cells and purified SV40 T antigen, the factors required for DNA replication and their mechanism of action will be studied. To date, seven cellular replication factors have been identified and partially characterized. These include replication factors A (RF-A) and C (RF-C), the Proliferating Cell Nuclear Antigen (PCN), DNA polymerases alpha and delta, and topoisomerases I and II. These replication factors will be further characterized and their role in DNA replication elucidated. Additional replication factors will also be identified and characterized. The proposed studies will determine the mechanism of initiation and elongation of DNA replication and may identify cellular proteins that are functional homologues of SV40 T antigen. These studies are directly relevant to the overall goals of this Program Project.